Promoter elements of the mouse 21-hydroxylase (Cyp-21) gene involved in cell-selective and cAMP-dependent gene expression

Cyp-21 (the mouse steroid 21-hydroxylase gene) is expressed exclusively in cells of the adrenal cortex, is induced by ACTH and cAMP, and is required for corticosteroid synthesis. This review examines the molecular basis for the regulated expression of Cyp-21 in the ACTH-responsive, mouse adrenocortical tumor cell line, Y1. We demonstrate that 330 bp of 5′-flanking DNA from the Cyp-21 gene are sufficient for cell-selective and ACTH-induced expression of Cyp-21, and that this promoter region comprises multiple, closely spaced enhancer elements each of which is required for promoter function. Within this promoter, we define three related elements that contain variations of an AGGTCA motif and that contribute to the cell-selective expression of Cyp-21. Variations of these same AGGTCA-bearing elements are also involved in the expression of Cyp 11a and Cyp 11b in Y1 adrenocortical cells. These elements interact with the same or closely related nuclear proteins found only in steroidogenic cell lines. Taken together, these results suggest that shared elements contribute to the adrenal cell-selective expression of at least three steroidogenic cytochrome P450 genes.

The element at −170 and the related elements at −65, −140 and −210 in the Cyp-21 promoter are not active as enhancers in the mutant Y1 cell line, Kin-8. Kin-8 cells contain a mutation in the regulatory subunit of the type 1 cAMP-dependent protein kinase that renders the enzyme resistant to activation by cAMP. Therefore, these elements appear to be selectively dependent upon an intact cAMP-dependent protein kinase for enhancer function. Individually, none of these elements confer cAMP-dependence to a reporter gene driven by a heterologous promoter. On the basis of these observations, we suggest that ACTH- and cAMP-dependent expression of Cyp-21 requires the combined actions of the element at −170, and the related elements at −140, −210 and −65.     

Chromosome-damaging effects of heraclenin in human lymphocytes in vitro

Heraclenin, a furocoumarin with an epoxide group in its side chain, was analyzed to see if it induced structural chromosome aberrations and sister-chromatid exchanges (SCEs) in human lymphocytes in vitro. The results were compared directly with those of imperatorin, which differs from heraclenin only in lacking an epoxide group. An equally strong clastogenic effect was found for both heraclenin and imperatorin: the number of metaphases with breaks was increased in both cases by approximately a factor of 6. Heraclenin produced a considerable dose-dependent increase in the SCE rate, i.e., by about 60 induced SCEs/metaphase, whereas imperatorin induced only about 4 SCEs/metaphase. The results are discussed with respect to the occurrence of structural aberrations, which are primarily due to the basic furocoumarin structure itself, whereas the large increase in the SCE rate produced by heraclenin is most probably significantly influenced by its epoxide group.     

Die Suppression der außerkaryotisch bedingten Streptomycin-Abhängigkeit bei Chlamydomonas reinhardii

Zusammenfassung Die Streptomycin-Abhängigkeit der Mutante sd₃ wird außerkaryotisch vererbt. Nach Übertragung auf streptomycin-freies Medium entstehen in einer Häufigkeit von 10^-7 bis 10^-6 streptomycin-empfindliche Zellen. Diese Revertanten unterscheiden sich in verschiedenen Merkmalen von dem ebenfalls streptomycin-sensiblen Wildtyp. Die Reversion kommt also nicht durch Rückmutation zustande. Durch Kreuzungsexperimente konnte gezeigt werden, daß die Mutation, die zur Suppression der Streptomycin-Abhängigkeit führt, auf der extranucleären DNS stattgefunden hat.

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Suppression of the extranuclear streptomycin dependence in Chlamydomonas reinhardii

Summary The streptomycin-dependent mutant sd₃ shows uniparental inheritance. From their transfer on streptomycin-free medium streptomycin-sensitive cells result in a frequency of 10^-7 to 10^-6. These revertants differ in several marks from likewise sensitive wild-type cells. Therefore they were formed not by backmutation. Crossings showed, that also this suppression took place on the extranuclear DNA.

     

Über die Zahl der Kopien eines außerkaryotischen Gens bei Chlamydomonas reinhardii

The streptomycin sensitive (ss-) revertants of the streptomycin dependent mutant sd₃ segregate on streptomycin medium again sd-cells. This sd-segregation decreases continuously with increasing time of cultivation until zero-level is reached. The revertant R 20 differ from the other revertants mainly that the rate of segregation remaining almost at the same high level a long time. But the results of clonings showed that also in R 20 the number of cells able to segregate sd-cells decreases. After 6 months, only 20% of the cells still possessed this ability. Former investigations showed that the ss-revertants still had sd-informations. These informations segregate in the following cell-divisions and produce again sd-colonies on the streptomycin medium. The rate of segregation was changing with the different clones. This proves the non-oriented distribution of the sd- and ss-gene copies at the mitotic division. The clonings demonstrate also the existence of much more than 2 copies. According to Sager and Ramanis the genes of the chloroplast exist at the most in 2 copies. If the statement should be true in principle for all vegetative cells, then the sd-information cannot be in the chloroplast. It must be located in the mitochondria.     

Clonal variation in adrenocorticotropin-induced desensitization of adenylate cyclase in Y1 adrenocortical tumor cells. Association with a 68,000-dalton protein

Adrenocorticotropin(ACTH)-induced desensitization of adenylate cyclase was examined in subclones derived from the ACTH-responsive, Y1 mouse adrenocortical tumor cell line. This report describes clonal variation in ACTH-induced desensitization of adenylate cyclase and an associated variation in the level of a 68,000-dalton protein, p68. A subclone of Y1 cells with a low level of p68 (0.8% of total protein) exhibited a faster rate of desensitization and a slower rate of recovery from desensitization when compared with a clone containing a high level of p68 (10% of total protein). In three clones with low levels of p68, ACTH desensitized adenylate cyclase with ED50 values from 0.3 to 0.5 nM. In several clones with high levels of p68, the adenylate cyclase system was more resistant to ACTH-induced desensitization; the ED50 values for ACTH in these clones ranged from 2 to 12 nM. Among 11 ACTH-responsive subclones, the level of p68 correlated significantly (p less than 0.001, r = 0.87) with resistance to the desensitization induced by 1 nM ACTH. These results suggest that p68 may function in the maintenance of an ACTH-responsive adenylate cyclase system, or that the level of p68 and responsiveness to ACTH are coordinately regulated.     

Induction of structural chromosome aberrations and sister chromatid exchanges in human lymphocytes in vitro by aristolochic acid

The medicinal use of Aristolochia clematitis has been known for some time. The main active agent of this medicinal plant is aristolochic acid, a nitrophenanthrenecarbonic acid. Very recently, however, the Federal Health Office withdrew the licence for all drugs containing aristolochic acid, because of the well-founded suspicion that aristolochic acid may be a very potent carcinogen. We investigated the induction of structural chromosome aberrations and sister chromatid exchanges (SCEs) by aristolochic acid in human lymphocytes in vitro. Cells were treated with the agent tested throughout culture time and during the G0 phase of the cell cycle. We tested concentrations over a range of 1 to 20 micrograms/ml. Both treatment conditions resulted in an increased aberration frequency. The induction of gaps and breaks as well as the induction of SCEs showed a dose-dependent increase. The number of SCEs per metaphase was enhanced by a factor of 2 to 3. If conventional cytogenetic methods had been applied in time, one would have recognized the mutagenic risk of aristolochic acid earlier.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.     

A 2,6,9-hetero-trisubstituted purine inhibitor exhibits potent biological effects against multiple myeloma cells

A focused library of hetero-trisubstituted purines was developed for improving the cell penetrating and biological efficacy of a series of anti-Stat3 protein inhibitors. From this SAR study, lead agent 22e was identified as being a promising inhibitor of MM tumour cells (IC₅₀’s <5 μM). Surprisingly, biophysical and biochemical characterization proved that 22e was not a Stat3 inhibitor. Initial screening against the kinome, prompted by the purine scaffold’s history for targeting ATP binding pockets, suggests possible targeting of the JAK family kinases, as well for ABL1 (nonphosphorylated F317L) and AAK1.